The present invention is directed to an apparatus and method for a near field scanning optical microscope, and more particularly optically recording near field images of living cells in an aqueous solution at a resolution higher than 50 nm, more than ten times greater than the wavelength of the light source used, and which provides a high resolution image and a rapid scan of the specimen (sample) without altering or damaging the specimen.
Far field optical microscopy, such as fluorescence microscopy, has become a major research tool in basic biomedical research and an effective diagnosis technique in clinical medicine. However, the need for high spatial resolution and signal sensitivity has so far severely limited the effectiveness of this far field method, and in fact, all optical microscopy related research. Similar limitations are also present when used in clinical applications. Presently, the far field technique has found broad applications in a number of areas, such as cancer cell detection, characterization of cell abnormalities and evaluation of fundamental processes involving the localization and function of membrane associated proteins, such as ion channels and surface receptors.
Unfortunately, further improvement in far field optical microscopy is limited by the diffraction limit of light. Currently available optical microscopes place the specimen on the image plane of the optical system (i.e., located at a distance far exceeding that of the wavelength). Yet diffraction limits the spot size formed in the case of a scanning beam type of microscope or the smallest features that can be resolved in the case of an imaging system. Therefore, the highest resolution of a conventional optical microscope is in the sub-xcexcm range, even when used with a confocal system.
Far field optical microscopy, regardless of the contrast mechanism being used (i.e., fluorescence, phase contrast or differential interference), is severely limited by the finite wavelength of light due to the unavoidable effect of diffraction. As such, the spatial resolution, either vertical to the plane of focus or lateral in the plane of focus, is roughly in the range of xcex/N.A., where xcex is the wavelength of the illumination (xcx9c0.5 xcexcm) and N.A. is the numerical aperture which can be up to 1.4. Higher resolution, along with improved signal collection efficiency, would significantly enhance the use of the optical technique.
To overcome this fundamental difficulty, a type of microscopy known as Near Field Scanning Optical Microscopy (NSOM or SNOM) technology has been developed. Rather than placing the specimen at a far distance from the light source, a near field scanning optical microscope (NSOM) places the specimen directly in front of the light source at a distance far smaller than the wavelength of light (i.e.,xe2x80x9cnear fieldxe2x80x9d). Since the physical size of the light source can be below the wavelength of the light, the spatial resolution would be limited by the size of the aperture. In this case, the illuminated area is no longer limited by diffraction. Therefore, by reducing the size of the light source (aperture), the volume being illuminated can be reduced accordingly. When such a light source is scanned over the specimen surface a two-dimensional image is obtained. Thus the system achieves the functions of a microscope. Since the resolution is directly related to the volume being illuminated, the resolution can be reduced to below that determined by diffraction.
Many techniques have been developed for fabrication of such small apertures. Normally a glass optic fiber can be pulled with laser heating to produce a very sharp apex, which can be coated with metal to make a small aperture at the end of the pulled fiber. Since only a very small volume is illuminated during the operating mode of a NSOM, a scanning mechanism must be used to acquire a two dimensional image. Since the image is obtained sequentially, the entire emission angle of 4xcfx80 is available for signal collection in the case of fluorescence imaging, thus improving the collection efficiency. This is an important practical factor to consider when taking into account the effects of photo-bleaching and frame-time for image acquisition.
To make a NSOM instrument useful, the position of the probe aperture must be precisely controlled to avoid crashing the probe into the specimen. It is also important to ensure the image contrast is due to fluorescence rather than variations of the distance between the probe and the specimen (sample) surface. When operated in air, distance control can be achieved using a vibrating probe sensitive to the probe sample interaction or other methods. As such, the NSOM technique and its variations do not satisfy the applications required by biomedical research and clinical medicine. This is because most biologically relevant applications need the specimen to remain fully hydrated, preserving its native structure and function. When a conventional NSOM probe is placed in aqueous solution, the viscosity of the media produces a much lower Q value in the vibrating device, rendering it completely insensitive to the minute probe-specimen interactions. NSOM has therefore had very little success when applied to biological systems.
For some NSOM devices, pulled optical fibers are used as a conduit for the light source. The optical fiber surface is coated with a thin layer of metal to render it optically opaque but the apex of the fiber remains open (acting as the aperture) to allow for the transmission of light. It has been possible to fabricate apertures as small as 10 nm. However, as mentioned above, a critical feature that is required to make the NSOM a practical instrument is the method of maintaining the position of the aperture at a fixed distance from the specimen surface during scanning. Otherwise, the surface topography will have a profound effect on image contrast, creating artifacts that cannot be separated from the optical information. Even though many schemes of controlling the probe position have been demonstrated, the most successful design thus far is based on shear force detection. Here, the probe is driven to laterally oscillate at its resonant frequency. When the aperture is near the specimen, the interaction between the probe and the specimen surface will produce a shift in the resonant frequency, leading to a change in the oscillation amplitude. Therefore, by locking on to a predetermined reduction in amplitude, the distance between the probe and specimen can be controlled. It is intended that the resulting optical signal is independent from surface topography when this separation distance can be precisely controlled. As mentioned earlier, the above-mentioned conventional NSOM devices do not work in solution.
As such, conventional NSOM technology has a number of drawbacks. As mentioned previously, a major application of optical microscopy is in the field of biology and biomedicine, including disease diagnosis and fundamental research. Most of these applications require that the specimen or the sample be completely immersed in aqueous environment in order to retain full specimen functionality. To the detriment of the conventional NSOM technologies, including the conventional shear force technique, when the optical probe is immersed in solution, the probe Q value (a value directly related to the sharpness of the resonance peak or quality of the resonance is diminished. Yet, the shear force technique detection requires a reasonably high Q value so as to prevent the probe from crashing into the specimen before a frequency or amplitude shift could be detected. This is a fundamental limitation of most mechanically based NSOM detection schemes. Therefore, truly high resolution NSOM imaging of biological specimens in aqueous solution have not been known despite many years of effort by numerous research groups and industrial laboratories. For these reasons, the NSOM technology has not had a major impact on the fields of biology and biomedical research. Without solving the above-mentioned technological limitations, the conventional NSOM approach will be ineffective in entering the main stream of biomedicine.
There is therefore a need in the art for an effective NSOM that provides a high resolution image of a specimen (sample) in an aqueous solution and a rapid scan(i.e., brief frame-time) of the specimen without altering or damaging the specimen.
According to the present invention, a near field scanning optical microscope (NSOM) comprises: a reservoir holding a sample to be scanned therein; a pipette having an open tip communicating with a hollow shaft; an electrolyte solution disposed within the reservoir covering the sample and disposed within the tip of the pipette; a first electrode disposed in the shaft in iconic communication with the electrolyte solution in the open tip, the first electrode being in ionic communication with electrolyte solution in the reservoir via the open tip by means of electrolyte solution within the tip; a second electrode disposed in the reservoir in ionic communication with the electrolyte solution in the reservoir and forming a continuous ionic current path between the first and second electrodes via the electrolyte solution in the reservoir and in the open tip, scanning means for scanning the tip of the pipette over a top surface of the sample in a scanning pattern; voltage means for applying a voltage across the first and second electrodes; current means for measuring a current flowing in the ionic current path between the first and second electrodes through the open tip of the pipette and for supplying an indication of the current at an output thereof; and control logic means having an output connected to the scanning means and an input connected to the output of the current means for causing the scanning means to set the height of the tip at a desired distance above the top surface. The present invention further comprises a light source disposed on the microscope for emitting light through the shaft of the pipette and onto the sample; the hollow shaft being opaque to substantially prevent light emitted from the light source from being transmitted through the walls of the shaft; and an image acquisition means in optical communication with the light source so that the sample is in optical communication between the light source and the image acquisition means, whereby the image acquisition means for acquiring an image of the sample.
Another aspect of the invention provides a method for optically imaging a sample comprising the steps of: disposing the sample to be scanned in a reservoir containing an electrolyte covering the sample; providing a pipette having an open tip communicating with a hollow shaft, wherein the shaft is at least partially opaque; disposing an electrolyte within the tip of the pipette; disposing a first electrode in the shaft in ionic communication with the electrolyte in the open tip; disposing a second electrode in the reservoir in ionic communication with the electrolyte in the reservoir and forming a continuous ionic current path between the first and second electrodes via the electrolyte solution in the reservoir and in the open tip; applying a voltage across the first and second electrodes and measuring an ionic current flowing in the ionic current path between the first and second electrodes through the open tip; scanning the tip of the pipette over a top surface of the sample in a scanning pattern with the tip of the pipette at a desired distance above the top surface which will maintain the current flow between the first and second electrodes through the open tip at a constant value which will cause the tip to follow the top surface in close non-contacting proximity thereto so as to provide a z-directional component of the position of the tip of the pipette; scanning the tip of the pipette over a top surface of the sample in a scanning pattern with the tip of the pipette in a plane parallel and close adjacent above the top surface; emitting light through the shaft of the pipette onto the sample; and acquiring light having been transmitted through the sample for acquiring an image of the sample and outputting a corresponding acquisition signal.